Sonic low pressure spray drying

ABSTRACT

This invention provides methods of spray drying pharmaceutical powders from a vibrating nozzle at low pressures. The method can effectively spray dry thick or viscous solutions or suspensions to provide small uniform particles. The invention includes dry particle compositions prepared by methods of low pressure spraying from vibrating nozzles.

This application is a Continuation application and claims benefit andpriority to U.S. application Ser. No. 12/266,493, which claims priorityto and benefit of a prior U.S. Provisional Application No. 61/002,308,Sonic Low Pressure Spray Drying, by Vu Truong-Le, et al., filed Nov. 7,2007. The full disclosure of the prior application is incorporatedherein by reference.

FIELD OF THE INVENTION

The invention is a method to stabilize pharmaceuticals using acombination of specific formulations and a spray drying processutilizing high frequency sonic or ultrasonic atomization nozzle. Inparticular, the methods provide preservation of viruses, bacteria andproteins prepared by low pressure high frequency spraying from vibratingnozzles.

BACKGROUND OF THE INVENTION

Successful stabilization of thermally labile biologicals requires acombination of appropriate formulations, a quick drying process withminimal thermal and mechanical stress, and solid state propertiesconducive to stable long term storage. Freeze drying and spray dryingare two of the most widely used methods for drying API solutions in thepharmaceutical industry. Spray drying provides advantages of high volumeproduct throughput (up to, e.g., 5,000 lb/hr) and reduced manufacturingtimes over batch process protein preservation/drying technologies, suchas freeze drying. The challenge of using spray drying to stabilizethermally labile APIs, such as biopharmaceuticals, involves the controlof four key areas: atomization conditions, drying conditions,formulation design, and resultant solid state properties of the driedmaterials. For example, during atomization, the process of breaking up aliquid stream into fine droplet sizes can involve excessive shearstress, surface tension, and pressure applied to the product, resultingin excessive loss of bioactivity. Additionally, some liquids exhibithigh viscosity and density, which requires higher atomization pressureresulting in overly broad droplet size distribution.

Most ultrasonic spray devices and techniques are directed to sprayingliquids to form uniform layers on surfaces or to provide high pressuresprays for fuel combustion. See, e.g., U.S. Pat. No. 4,978,067, UnitaryAxial Flow Tube Ultrasonic Atomizer with Enhanced Sealing, or U.S. Pat.No. 4,541,564, Ultrasonic Liquid Atomizer, Particularly for High VolumeFlow Rates, to Berger, et al. However the problems solved by thesetechniques are different from those in spray drying of pharmaceuticals.Ultrasonic spray drying of bioactive materials demands high dropletuniformity, quick drying and low stress not found in the prior art.

Conventional ultrasonic nozzles use a piezoelectric transducer toconvert electrical energy to mechanical vibrations at ultrasonicfrequency range (i.e. greater than 20 kHz). The ultrasonic vibrationsare focused at the tip where, as the liquid flows through, theoscillating tip disintegrates the liquid into micro-droplets, and ejectsthem to form a gentle, low velocity spray of freely flowing formulationsat ambient pressure (i.e., pressure-less) conditions. Because the mainenergy source controlling atomization is mechanical vibrations, thedroplet size distribution of the atomized liquid is primarily a functionof frequency, and the higher the frequency, the smaller the dropletdiameter. However, typical median droplet size of aqueous fluids usingthese techniques is ˜90 microns at 20 kHz, and 45 microns at 40 kHz,which are still large for efficient, fast drying of the droplets to formdry stabilized powders. Another limitation is that the higher thefrequency, the lower the processing capacity (i.e. flow rate).

In view of the above, a need exists for a method to spray thickpharmaceutical and vaccine formulations under low shear stressconditions. It would be desirable to be able to spray formulations witha wide range of solution concentrations under low pressures whileproviding small droplets of uniform size. The present invention providesthese and other features that will be apparent upon review of thefollowing.

SUMMARY OF THE INVENTION

The methods of the invention include spraying a solution or suspensionof a bioactive material flowing at low pressure through a vibratingnozzle. For example, the methods of preparing pharmaceutical powderscontaining bioactive materials can include the steps of preparing asuspension or solution comprising the bioactive materials and at least20% hydrophilic solids by weight, forming a mixture of a pressurized gaswith the solution or suspension within a nozzle, vibrating the nozzle ata high vibration frequency ranging from 1 kHz to 100 kHz while sprayingthe mixture to form a gaseous suspension of droplets, wherein the highfrequency vibrations are generated using the pressurized gas, and dryingthe droplets in a stream of drying gas to form powder particles of thebioactive material. Admixture of a spray gas for high frequencyvibration spraying provides a spray different in character, especiallyin combination with thick formulations. Further differences are realizedwhen the working gas creating the vibrations is also present in theflowing stream from the nozzle. Using the above described methods,particles ranging in average size from about 1 μm to about 20 μm can besprayed from solutions at pressures lower than 50 psi, even where thesolution is thick or viscous, e.g. solids content up to 70% w/v.

The bioactive material for preservation in the pharmaceutical powderscan include, e.g., bioactive proteins, peptides, antibodies, enzymes,serums, vaccines, nucleic acids, bacteria, prokaryotic cells, eukaryoticcells, liposomes, viruses, and/or the like. Preferred viruses forinclusion in the powders can include, e.g., rotavirus, adenovirus,measles virus, mumps virus, rubella virus, polio virus, influenza virus,parainfluenza virus, respiratory syncytial virus, herpes simplex virus,Severe Acute Respiratory Syndrome (SARS) virus, corona virus familymembers, cytomegalovirus, human metapneumovirus, filovirus andEpstein-Bar virus. Preferred bacteria for spraying into the powders caninclude, e.g., Pneumococcus, Lactobacillus, Francisella tularensis,Mycobacterium, Salmonella, Shigella, Listeria, Pseudomonas,Staphylococcus, Streptococcus, and E. Coli.

The suspension or solution for spraying can be quite viscous or thick.For example, the sprayed solution or suspension can include substantialamounts of sugars and/or polymers. In preferred embodiments, thesolution or suspension includes two or more sugars totaling 25% or moreof the suspension or solution by weight. Preferred sugars or polyols canbe, e.g., sucrose, trehalose, glucose, raffinose, sorbose, melezitose,glycerol, fructose, mannose, maltose, lactose, arabinose, xylose,ribose, rhamnose, galactose, glucose, mannitol, xylitol, erythritol,threitol, dextrose, fucose, trehalose, polyaspartic acid, inositolhexaphosphate (phytic acid), sialic acid, N-acetylneuraminicacid-lactose, and sorbitol. Preferred polymers can include, e.g.,polyvinyl pyrrolidone (PVP), gelatin, collagen, chondroitin sulfate,starch, starch derivatives, carboxymethyl starch, hydroxyethyl starch(HES), polyvinyl alcohol, and/or dextran.

The solutions or suspensions can include other excipients, such assurface active agents, amino acids, and divalent cations. For example,the solutions or suspensions can include a surfactant, such as a blockcopolymer of polyethylene and polypropylene glycol, polyethylene glycolsorbitan monolaurate, and polyoxyethylene sorbitan monooleate. Preferredamino acids include, e.g., arginine, alanine, lysine, methionine,histidine, and glutamic acid. Examples of divalent cations include,e.g., Ca²⁺, Zn²⁺, Mg²⁺, and Mn²⁺.

The solution or suspension can be mixed with a low pressure gas, suchas, e.g., air, nitrogen, oxygen, helium, carbon dioxide, sulfurhexafluoride, chlorofluorocarbons, methane, fluorocarbons, nitrousoxide, xenon, propane, n-pentane, ethanol, water, or the like. Formingthe mixture can be accomplished by flowing the solution or suspensionwith the pressurized gas within a mixing chamber, e.g., within thenozzle. Typically, the mixing occurs before the mixture components exitthe spraying nozzle. The mixture can be formed by flowing the suspensioninto the nozzle at a pressure less than 10 psi, and flowing the gas intothe nozzle at a pressure between 10 psi and 50 psi. In preferredembodiments, the pressurized gas is characterized by a pressure of about50 psi or less. In preferred embodiments, the pressurized gas ischaracterized by a temperature ranging from about 0° C. to about 90° C.

In certain embodiments, the gas and/or solution includes a modifier,such as, e.g., methanol, ethanol, isopropanol, chloroform, heptane,methyl isobutyl ketone, tetrahydrofuran, ethyl acetate, dichloromethane,dichloromethane:ethanol:isopropanol (5:6:4), acetone and/or the like.

The vibrations of the nozzle can help provide smaller spray dropletsthan would otherwise occur under the conditions without nozzlevibrations. The nozzle can be vibrated by input of energy, e.g., byinput of electrical energy, or the energy of a pressure differential.For example, the vibrations can be due to provision of pulsatingvoltages to a piezoelectric crystal, or by resonance of fluids flowingwith a pressure differential between the suspension or solution and thepressurized gas within the nozzle. The vibration can be a “whistling”resonance of a high pressure gas entering a lower pressure chamberthrough an appropriately configured orifice.

The nozzle can beneficially vibrate at a high vibration frequencyranging through the sonic range and into the ultrasonic range. Forexample the vibration can range from about 20 Hz to 200 kHz, from 100 Hzto 150 kHz, from 1 kHz to 100 kHz, from 10 kHz to 50 kHz or about 20kHz. In a preferred embodiment, the high frequency vibrations range fromabout 10 kHz to about 30 kHz.

In certain embodiments, the spray dried powders can be furtherstabilized, e.g., by suspending them in a hydrophobic liquid. This canhelp exclude damaging water and oxidants from the bioactive material. Insome embodiments of the invention, the methods further comprisesuspending the powder particles in an organic solvent. Further, highlystable or time releasable forms of the bioactive material can beprovided by drying the solvent to form a solid material comprising thebioactive material. In preferred embodiments, the dry particles of thebioactive material include a polymer with some hydrophobic character. Inpreferred embodiments, the bioactive material is not denatured when thepowder particles are suspended in the organic solvent.

The present invention includes compositions of powder particles withunique properties, e.g., produced using the methods of the invention.The compositions can include spray dried particles comprising abioactive material in a glassy matrix of two or more sugars wherein thebioactive material comprises less than 10% or less than 1% of theparticle by weight, and wherein the particles were spray dried in thepresence of ultrasonic vibrations from a solution or suspensioncomprising at least 20% total of the two or more sugars. The powderparticles can optionally be suspended in an organic solvent. In a morepreferred embodiment, the composition of powder particles includes twoor more sugars, such as, e.g., sucrose and trehalose, and the two ormore sugars comprise 25% to 50% of the suspension or solution by weight.In a most preferred embodiment, the suspendion or solution for highfrequency vibration spraying includes from 7% to 28% w/v sucrose, from3% to 12% w/v trehalose, from 25 mM to 100 mM potassium phosphatebuffer, from 0.5 mM to 20 mM of a divalent cation, from 0.25% to 1% v/vof a plasticizer, and from 0.02% to 0.08% v/v of a surfactant.Plasticizers can include those known in the art such as, e.g., DMSO,glycerol and sorbitol.

DEFINITIONS

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein havemeanings commonly understood by those of ordinary skill in the art towhich the present invention belongs.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular devices orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acomponent” can include a combination of two or more components;reference to “a buffer” can include mixtures of buffers, and the like.

Although many methods and materials similar, modified, or equivalent tothose described herein can be used in the practice of the presentinvention without undue experimentation, the preferred materials andmethods are described herein. In describing and claiming the presentinvention, the following terminology will be used in accordance with thedefinitions set out below.

The term “about”, as used herein, indicates the value of a givenquantity can include quantities ranging within 10% of the stated value,or optionally within 5% of the value, or in some embodiments within 1%of the value.

The term “low pressure gas”, as used herein, refers to gasses atpressures of less than 200 psig, less than 100 psig, less than 50 psig,less than 25 psig, less than 10 psig, or less. Low pressure gasses areat pressures more than 10% below, typically more than 20% below, theircritical pressure at a given temperature. Although a higher pressure gasmay be introduced to a mixing chamber of a nozzle, the spraying can beconsidered low pressure based on the pressure within the chamber.

The term “ultrasonic”, as used herein, indicates a frequency above 20kHz. With regard to the present invention, preferred ultrasonicfrequencies for vibration of a spray nozzle can be from about 20 kHz toabout 200 kHz, form about 40 kHz to about 150 kHz, from about 50 kHz toabout 100 kHz, or about 75 kHz. The term “high frequency” refers to highsonic pitches (e.g., above about 1 kHz) and ultrasonic frequencies.

As used herein, the terms “solution and suspension” and “liquidformulation” are often used interchangeably.

A bioactive material, such as an enzyme, antibody, vaccine antigen,virus, bacteria, protein receptor, nucleic acid, and the like, has abioactivity, as understood in the art.

Hydrophilic solids are molecular species that exist as a solid at roomtemperature and are soluble at concentration of at least 10 mM inaqueous solution at 25° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of an exemplary high frequency spraynozzle.

FIG. 2 shows a chart of particle size distribution for spraying 10%sucrose at a pressure of 20 psi from a nozzle vibrating at a highfrequency.

FIG. 3 shows a chart of particle size distribution for spraying 10%sucrose at a pressure of 30 psi from a nozzle vibrating at a highfrequency.

FIG. 4 shows a chart of particle size distribution for spraying water ata pressure of 20 psi from a nozzle vibrating at a high frequency.

FIG. 5 shows a chart of stability for sonic spray-dried particlescontaining viral bioactive material.

FIG. 6 shows a chart of Salmonella viability before and after spraydrying using methods of the invention.

FIG. 7 is a chart showing stability data for storage of sonic spraydried Salmonella.

DETAILED DESCRIPTION

The present invention is directed to methods of spray drying that canspray high solids concentration solutions or suspensions for quickdrying without the use of high pressures. The application of the powderproduction methods provides advantages of high volume productthroughput, reduced manufacturing times over other pharmaceutical dryingmethods such as lyophilization. The presently described ultrasonic spraydrying methods are broadly applicable to all pharmaceutical compoundsand can achieve stabilization better than spray drying methods employingconventional two-fluid nozzles.

In typical embodiments of the inventive methods, a liquid formulation ofa bioactive material is mixed with a stream of low pressure gas. Themixture is exposed to high frequency vibrations and sprayed as dropletsinto a drying chamber. A drying gas removes liquid solvents from thedroplets leaving dry powder particles. The technique has the advantagesof being able to spray thick solutions or relatively coarse particulatesuspensions without exposing them to harmful levels of pressure, shear,processing time, and temperature. The combination of low pressurespraying and vibration treatment can provide more uniform droplets fromeven hard to spray liquids. The thick and/or uniform droplets can bedried more quickly and/or at lower temperatures.

Key attributes to this invention involve the identification of uniqueformulation combinations well suited to application of low pressureultrasonic nozzles, such as the SONICAIR™ nozzle (IVEK corp., N.Springfield, Vt.).

As the solids content is increased, the liquid load to be drieddecreases, and hence higher processing throughput is achieved.Additionally, the combination can provide a smaller droplet sizedistribution, and a higher surface area-to-volume ratio, which meansfaster drying at lower heating load. Lower heat load exposes thebioactive material to a lower thermal stress, thereby reducingprocess-related bioactivity loss. Therefore, the application of a moreoptimized nozzle can reduce the stresses involved in atomization anddrying. Further stress reduction and improvement in storage stabilityinvolves rational selection of stabilizing excipients. Formulatedprotein powders can include other substances to enhance activestability, reduce process losses and reduce reconstitution times, suchpolyhydroxy compounds (sugars), amino acids, divalent cations,surfactants, and other water soluble polymers (synthetic or naturallyderived).

In the present invention low pressure sprayed high frequency atomizedspray dried powders were found to be surprisingly stable suspended in avariety of organic solvents (or solvent mixtures) for up to one hour at55° C. This property allowed the active proteinaceous ingredient such asantibodies, enzymes, bacteria, viruses, cells, etc to remainbiologically active under manufacturing processes involving harshorganic solvents and heat. For example, this stabilizing propertyenabled the incorporation of formulated, spray dried live viruses intowafers or thin films for oral delivery. Excipients competing withbioactive proteins for the water/organic solvent interface surfactants(e.g. polyoxymers, Tween, etc.) and polymers (e.g. polyvinyl alcohol,etc) can help protect the proteins against exposure to organic solventsby driving the proteins into the core of the particles and away from theparticle surface. Moreover, excipients that exhibit preferentialhydrogen bonding or ‘hydration’ of the protein in the aqueous and in thedry state, such as sucrose, may stabilize the protein via watersubstitution yielding a protective coating around the protein surface.[Cleland, J L and Jones, A J. Pharm Res. 1996 October; 13(10):1464-75].

Applicable active pharmaceutical compounds for preservation by theinventive methods include, e.g., monoclonal antibodies, therapeuticproteins, peptides, live viruses, live bacteria, and other bioactivematerials. The stabilizing formulation components include, but are notlimited to, a buffer, a polyol, a surfactant, a plasticizer, an aminoacid, and/or a polymer. The spray drying process can employ highfrequency atomization nozzles to produce dry powder particles that canbe room temperature stable and exhibit appropriate powder properties fordeep lung delivery as well as for fabrication into other dosage formatssuch as oral wafers, oral thin films, capsules, tablets, etc.

In an exemplary embodiment of the inventive methods, a solution ofactive pharmaceutical ingredient (API) is first formulated withstabilizing excipients, then atomized from a sonic nozzle or ultrasonicnozzle operated at a low pressure range (5-100 psig) using a pressurizedgas, with or without an organic solvent serving as a liquid modifier.The atomized API is caused to dry into powder particles by infusing astream of dry, heated gas co-current to the spray plume. The spraydrying equipment can be any commercially available spray dryers butsubstituted with commercially available ultrasonic nozzles. Preferablythe sonic/ultrasonic nozzles are non-piezoelectric and operate at lowpressure range. The atomizing gas can be air or any other gases,preferably air, nitrogen, CO₂ at or near supercritical state.Preferably, the atomizing gas is introduced to a liquid stream of theformulated API before the ultrasonic spraying step. The gas used toevaporate the atomized solution, i.e. the drying gas, is typicallyheated and can be air, nitrogen, argon, or the like.

Preparing a Solution or Suspension

In the present invention, solutions or suspensions can be prepared usingappropriate techniques known in the art. Appropriate formulations forinput to the low pressure vibrating spray dry processes of the presentinvention can, e.g., include relatively fragile bioactive materials,include relatively thick (viscous) solutions or suspensions, and/orinclude relatively high total solids content. Formulations for sprayingin the present invention include, e.g., one or more desired bioactivematerials plus one or more excipients.

Bioactive materials in the formulations for spraying in the presentinvention include, e.g., bioactive proteins, nucleic acids, livingcells, viruses, bacteria, fungi, protista, and/or the like. Excipientconstituents of the formulations can include sugars, polymers, buffers,surfactants, chelators, salts, amino acids, and/or the like.

Bioactive protein materials can include any enzymes, ligands,antibodies, antigens, receptors, cytokines, active fragments, and/or thelike. For example, bioactive proteins can include, but are not limitedto, growth factors, cytokines, antigens, antibodies, interleukins,lymphokines, interferons, enzymes, etc., including, but not limited to,anti-IgE antibodies, tissue plasminogen activator (tPA), calcitonin,erythropoietin (EPO), factor IX, granulocyte colony stimulating factor(G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF),growth hormone (particularly human growth hormone), heparin (includinglow molecular weight heparin), insulin, insulin-like growth factors I(IGF-I) and II (IGF-II), interleukins, α-interferon, β-interferon,γ-interferon, luteinizing hormone-releasing hormone, somatostatin andanalogs, vasopressin and analogs, follicle stimulating hormone, amylin,ciliary neurotrophic factor, growth hormone releasing factor,insulinotropin, macrophage colony stimulating factor (M-CSF), nervegrowth factor, parathyroid hormone, α-1 antitrypsin, anti-RSV(respiratory syncytial virus) antibody, DNase, Her2 (Human Epidermalgrowth factor Receptor 2) and the like. Viruses in the formulation caninclude, e.g., influenza virus, parainfluenza virus, respiratorysyncytial virus, human metapneumovirus, corona virus family members,human immunodeficiency virus, herpes simplex virus, cytomegalovirus,SARS (Severe Acute Respiratory Syndrome) virus, Epstein-Barr virus,and/or the like. Bacteria in the formulation can include, e.g.,Salmonella, Shigella, Listeria, Lactobacillus, Pseudomonas,Staphylococcus, Streptococcus, and E. Coli.

Sugar excipients can include, e.g., monosaccharides (galactose,D-mannose, sorbose, etc.), disaccharides (lactose, trehalose, sucrose,etc.), cyclo dextrins, and polysaccharides (raffinose, maltodextrins,dextrans, etc.). Preferred sugars are, e.g., sucrose, trehalose,glucose, raffinose, sorbose, melezitose, glycerol, fructose, mannose,maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose,glucose, mannitol, xylitol, erythritol, threitol, and sorbitol. Inpreferred embodiments, sugars are the major excipient constituent of theformulations (solvents not being considered excipients). In manyembodiments of the methods, sugars account for 5% to 99% of the totalsolids, 20% to 90%, 30% to 80%, 50% to 70% or 60% of the total solids.In many embodiments, the liquid formulation for spraying includes fromabout 5% to about 70% sugar by weight, 20% to 60%, 30% to 50% or about40% sugar by weight.

Polymers can be useful, e.g., in certain time-release compositions, informing protective matrices around bioactive materials and/or to providestructural strength to sprayed products. Exemplary polymers useful inthe formulations can include carrageenan, polylactides, copolymers ofL-glutamic acid and gamma-ethyl-L-glutamate, human serum albumin (HSA),nonhydrolyzed gelatin, poly(2-hydroxyethyl methacrylate), ethylene vinylacetate, poly-D-(−)-3-hydroxybutyric acid, actin, dextran, myosin,methylcellulose, xanthan gum, polyvinyl pyrrolidone, collagen,chondroitin sulfate, a sialated polysaccharide, microtubules, dynein,kinetin, hydrolyzed gelatin, and/or the like.

Buffers can be useful in enhancing the stability of many bioactivematerials. Suitable buffers include, e.g., acetate, citrate, succinate,ammonium bicarbonate, phosphate, carbonate, and the like. Generally,buffers are used at molarities from about 1 mM to about 1 M. Typically,the buffers are selected and adjusted to provide a physiological pH,such as pH 7.4, of a pH ranging from about pH 4 to about pH 9, from pH 5to pH 8, or about pH 7.

Surfactants can be included in the formulations, e.g., to stabilizemembranes, help reduce droplet sizes, enhance the solubility of otherformulation constituents, and the like. Preferred surfactants in theformulations are typically non-ionic surfactants. For example, preferredsurfactants can include block copolymers of polyethylene andpolypropylene glycol, polyethylene glycol, sorbitan monolaurate, andpolyoxyethylene sorbitan monooleate.

Salts and/or chelators can be included in the formulations, e.g., tohelp stabilize protein structures, viral capsids, cell membranes.Optionally, removal of certain metal ions with chelators can reducedegradation due to proteases, nucleases, lipases and the like. Exemplarysalts and chelators can include, e.g., Ca²⁺, Zn²⁺, Mg²⁺, and EDTA.

Certain zwitterions can be useful in stabilizing the formulations.Formulations used in the methods often include one or more amino acids,such as, e.g., arginine, alanine, lysine, methionine, histidine, andglutamic acid.

A convenient way to prepare a solution of suspension formulation forspraying in the recent methods is to harvest and/or purify the desiredbioactive material in a somewhat concentrated form, then to mix thebioactive material with a concentrated stock formulation in appropriateproportions. For example, a centrifuged pellet of bacterial cells orvirus can be blended into a 1.5× formulation stock, and sufficientquantity of pure water added to obtain a 1× formulation. Alternately, a2× concentrate of a bioactive molecule of cell can be mixed 1:1 with a2× formulation concentrate. Optionally, the desired amount of bioactivematerial can be dialyzed into a desired volume of a 1× formulationstock.

Forming a Mixture with a Low Pressure Gas

In the present methods, the formulation is typically mixed with a gas,at a low pressure before or during exposure to ultrasonic vibrations.Although the formulation and/or gas can be delivered to the proximity ofthe mixing chamber at relatively high pressures, it is preferred theblending of the spray gas and formulation takes place at relatively lowpressures.

Often the mixture of liquid formulation and low pressure gas takes placein a mixing chamber. The mixing chamber can be simply a conduit throughwhich the formulation and gas are made to flow through together.Optionally, the mixing chamber can include obstructions, textures,baffles, etc., that enhance the intermixing of the gas and liquid phasesas they flow through the chamber. Optionally, there is no substantialmixing chamber, but, e.g., only a common port where the formulation andgas exit a vibration nozzle together.

The spray gas and liquid formulation can be mixed in a controlled massflow ratio. The mass flow ratio (gas/liquid) of the low pressure gasflow rate to the suspension or solution flow rate can be between about0.1 and 100, preferably between 1 and 20, more preferably between 1 and10, and most preferably between 0.5 and 2. In many embodiments the massflow ratio is about 1. Because the ultrasonic nozzle helps reduce thesize of droplets formed in the methods, relatively low gas flow ratescan be adequate compared to other spray dry techniques.

Pressures within mixing chambers typically range from about 5 psig toabout 100 psig. Although the gas and/or formulation may be delivered tothe mixing chamber of ultrasonic nozzle at higher pressures, they aretypically stepped down, e.g., by passage through a constriction,resonator, valve or diaphragm before the mixing or spraying steps. Themixture typically takes place at a low pressure ranging, e.g., fromabout 3 psig to about 100 psig, from about 5 psig to about 75 psig, fromabout 10 psig to about 50 psig, or about 30 pisg.

Vibrating a Spray Nozzle

It is an aspect of the present invention that mixtures of spraying gasand liquid formulations of bioactive materials are exposed to highfrequency vibrations (e.g., high sonic or low ultrasonic frequencies)before the mixtures are sprayed for drying. The mixtures can be vibratedat high sonic frequency or “sonicated” at ultrasonic frequencies, e.g.,as the mixture is being formed (e.g., in a mixing chamber), within thebody of a sonic nozzle, and/or at an exit orifice of a sonic nozzle.Optionally, the mixtures can be formed before exposure to the highfrequency vibrations.

Vibrating a spray nozzle at a high frequency can be accomplished usingany technique known or practiced in the art. For example, as shown inFIG. 1, it is known to vibrate spray nozzles at ultrasonic frequenciesby contacting the nozzle with a piezoelectric device 10 configured tovibrate at an ultrasonic frequency. Piezoelectric actuators 11 can beincorporated into the body of a spray nozzle and energized with electricvoltage at an ultrasonic frequency to vibrate the nozzle along the axisof the nozzle and/or in a direction lateral to the nozzle axis.

Alternately, sonic or ultrasonic vibrations can be generated in a nozzleby the harmonic interaction of a pressurized fluid passing through aconduit or port. For example, sonic nozzles can be activated bypressurized gas or fluid passing through a resonance chamber, e.g.,passage through a convergent/divergent section of a conduit at highvelocities to expand into a resonator cavity where it is reflected backto complement and amplify a primary shock wave (e.g., similar infunction to a sonic toy whistle). The result can be an intensified fieldof sonic energy focused between the nozzle body and the resonator. Thefrequency of the resonance can be influenced, e.g., by the mass of thehigh pressure fluid, the fluid pressure, the size of the resonatorcavity, the mass of the nozzle, the location of the resonator cavitywithin a nozzle, etc.

The size and uniformity of the droplets thus sprayed can be influencedby the vibrations. For example, the frequency, amplitude and directionof the vibrations can affect the droplets. In general, median dropletsize is inversely proportional to frequency to the ⅔ power, such that ahigher frequency will provide smaller droplet size; and a higher powerwill provide a smaller droplet size, up to a point. Of course, resultscan vary, e.g., in combination with other droplet size-affectingparameters, such as liquid flow rate, liquid viscosity, mass ratio,mixture pressure, etc.

In preferred embodiments, the mixture is exposed to spray nozzleoperating at high sonic to ultrasonic frequencies ranging from 1000 Hzto 200 kHz. In many embodiments useful for spraying bioactiveformulations, the preferred frequencies range from about 5 kHz to about100 kHz, from 10 kHz to about 70 kHz, or from 30 kHz to about 50 kHz. Inmany embodiments, the preferred nozzle vibration frequency ranges fromabout 10 kHz to about 30 kHz.

For a typical liquid formulation flow of 1 ml/min, the sonic/ultrasonicnozzle energy can range, e.g., from 0.1 W to 20 W, from 0.5 W to 10 W,or about 1 W.

Sonicating nozzles typically vibrate with a sin wave, which may bepolarized, or not. Optionally, the vibrations, in any dimension, candescribe a saw tooth, square wave, clipped wave, etc. The vibrations canbe along the axis of the nozzle (e.g., spray direction), across theaxis, and/or around the axis.

Drying Droplets

Droplets of suspensions or solutions can be dried to form particles. Thedrying can be by any means appropriate to the droplet composition andintended use. For example, the droplets can be sprayed into a stream ofdrying gas, onto a drying surface, into a cold fluid to freeze thedroplets for later lyophilization, and/or the like. “Dry” particles aretypically not liquid and can have a moisture content (e.g., residualmoisture) of less than 15%, less than 10%, less than 5%, less than 3%,less than 1.5% or about 1%.

In one embodiment, the droplets are sprayed into a stream of a dryinggas. For example, the drying gas can be an inert gas, such as nitrogen,at a temperature ranging from ambient temperatures to 200° C. Becausethe present methods can provide droplets of small uniform size, high insolids, drying conditions can be moderate, thus minimizing stress anddegradation of constituent bioactive materials. In many cases, thestream of drying gas can enter the drying chamber to contact thedroplets at a temperature of 150° C. or less, 100° C., 70° C., 50° C.,30° C. or less. The particles can be collected by settling, filtration,impact, etc. Particles can be exposed to secondary drying conditions toremove additional moisture.

Alternately, the droplets can be lyophilized to dryness. In oneembodiment, the droplets are sprayed into liquid nitrogen to form frozendroplets. The droplets can settle out of the liquid nitrogen, or beremoved by filtration or evaporation of the nitrogen. The collectedfrozen droplets can be placed in a vacuum chamber and lyophilized toform dry particles, e.g., without ever exposing the bioactive materialsto high temperatures.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Low Pressure Ultrasonic Spray Drying

Various formulations and process conditions were used to spray dryviruses. In each case, the spray technique involved a combination of thecited formulation with a spray gas at low pressure. The gas/liquidmixture was then sprayed from a nozzle energized with ultrasonicvibrations.

Study 1—

A live G3 strain type rotavirus was low pressure sonically spray driedunder the following conditions:

a) A liquid formulation of rotavirus was blended with a concentrate ofSD01 formulation (1×: 7% w/v sucrose, 3% w/v trehalose, 25 mM potassiumphosphate buffer, 0.25% v/v glycerol, 0.02% v/v Pluronic F-68, pH 7.0)to provide a solution or suspension comprised of 6.87 log ffu/mL(fluorescence focus units/mL) rotavirus and 25% solids content.

b) The formulation was combined at a flow of 0.75 mL/min with a streamof nitrogen gas at 40 psi in a mixing chamber of a nozzle.

c) The nozzle was vibrated at ultrasonic frequencies.

d) The formulation/gas mixture was sprayed into a drying chamber whiledrying gas flowed into the chamber at 70° C. Drying gas exited thechamber at 50° C. outlet temperature.

e) Dry particles containing 2.45% residual moisture were collected.After reconstitution, the formulation had a time zero (t₀) titer of 6.94log ffu/mL (no significant loss of virus viability).

f) To study the stability of the spray dried rotavirus, dry powderparticles were held at elevated temperatures and the virus titer wasmonitored. After 6 days at 37° C., the rotavirus had a reconstitutedtiter of 6.68 log ffu/mL. After 6 days at 45° C., the rotavirus had areconstituted titer of 6.73 log ffu/mL.

Study 2—

A live G3 strain type rotavirus was low pressure sonically spray driedunder the conditions described above, with the exception of adjustingthe drying gas inlet temperature to 50° C. and the outlet temperature to40° C. The resultant powder particles were dried to a moisture contentof 3.51% and retained a reconstituted titer of 6.62 log ffu/mL.

Study 3—

A live G3 strain type rotavirus was low pressure sonically spray driedunder the conditions described in Study 1, with the exception ofadjusting the spray gas pressure to 60 psi, the drying gas inlettemperature to 50° C., and the outlet temperature to 40° C. Theresultant powder particles were dried to a moisture content of 2.43% andretained a reconstituted titer of 6.54 log ffu/mL.

Study 4—

A live G3 strain type rotavirus was low pressure sonically spray driedunder the conditions described in Study 1, with the exception ofadjusting the spray gas pressure to 60 psi, the drying gas inlettemperature to 50° C., and the outlet temperature to 40° C. A liquidformulation of rotavirus was blended with a concentrate of SD03formulation (1×: 7% w/v sucrose, 3% w/v trehalose, 25 mM potassiumphosphate buffer, 0.02% v/v Pluronic F-68, pH 7.0) to provide a solutionor suspension comprised of 6.82 log ffu/mL and 25% solids content. Theformulation was low pressure ultrasonically spray dried (to 4.29%moisture), as described above, followed by freeze drying for 16 hours at4° C. (to 2.66% moisture). The resultant powder particles retained areconstituted titer of 6.57 log ffu/mL.

Study 5—

A live G3 strain type rotavirus was low pressure sonically spray driedunder the conditions described in Study 1, with the exception ofadjusting the spray gas pressure to 60 psi, the drying gas inlettemperature to 50° C., and the outlet temperature to 40° C. A liquidformulation of rotavirus was prepared with a concentrate of SD02formulation (1×: 7% w/v sucrose, 3% w/v trehalose, 25 mM potassiumphosphate buffer, 0.5% glycerol, 0.02% v/v Pluronic F-68, pH 7.0) toprovide a solution or suspension comprised of 6.77 log ffu/mL rotavirusand 25% solids content. The formulation was low pressure ultrasonicallyspray dried (to 3.33% moisture), as described above, followed by freezedrying for 16 hours at 4° C. (to 2.26% moisture). The resultant powderparticles retained a reconstituted titer of 6.37 log ffu/mL.

Study 6—

Spray drying live rotavirus G3 vaccine at high solids content. A live G3type rotavirus was low pressure sonically spray dried under theconditions described in Study 1, with the exception of adjusting thespray gas pressure to 60 psi, the drying gas inlet temperature to 50°C., and the outlet temperature to 40° C. The virus was formulated withliquid formulation SD01, however, providing a total solids content of50%; see FIG. 5, Stability of spray dried G3 rotavirus at 37° C. and 45°C. storage.

Study 7—

A live G1 strain rotavirus was low pressure sonically spray dried underthe conditions described in Study 1, with the exception of adjusting thespray gas pressure to 15 psi, the flow rate to 0.5 mL/min, the dryinggas inlet temperature to 60° C., and the outlet temperature to 40° C.The virus was titrated to 6.2 log ffu/mL and formulated with liquidformulation SD01 (at 20% solids content) containing three additionalcomponents, which were 2 mM ZnCl₂, 2 mM CaCl₂, and 0.8% (w/w) sodiumcitrate. The solution pH was adjusted to 6.3. This formulation isreferred to as SD01ZC. Process loss was less than 0.2 log ffu/mL and theinitial titer did not decrease after 2 months of storage at 37° C., orafter 3 months of storage at 4° C. and at 25° C. The spray dried powdercontained 2.4% residual moisture content.

Study 8—

A live G1 strain rotavirus was low pressure sonically spray dried underthe conditions described in Study 1, with the exception of adjusting thespray gas pressure to 15 psi, the flow rate to 0.5 mL/min, the dryinggas inlet temperature to 60° C., and the outlet temperature to 40° C.The virus was titrated to 6.2 log ffu/mL and formulated with 21.6% (w/v)sucrose, 33 mM potassium phosphate, 15 mM glutamate, 2 mM ZnCl₂, 2 mMCaCl₂, and 0.8% (w/w) sodium citrate. The solution pH was adjusted to6.3. Process loss was negligible, however the initial titer decreased by1.2 log ffu/mL after 2 months of storage at 37° C. The titer decreasewas much lower after 3 months of storage at 4° C. and at 25° C. (<0.1log ffu/mL). The spray dried powder contained 3.1% residual moisturecontent.

Study 9—

A mixture of live G1 and G3 type rotavirus was spray dried using anultrasonic nozzle at low pressure under the conditions described inStudy 1, with the exception of adjusting the spray gas pressure to 15psi, the flow rate to 0.5 mL/min, the drying gas inlet temperature to60° C., and the outlet temperature to 40° C. Both of the strains weretitrated to 6.1 log ffu/mL and formulated with SD01ZC. The solution pHwas adjusted to 6.3.

Study 10—

A mixture of live G1, G3, G4, and G9 type rotavirus was spray driedusing an ultrasonic nozzle at low pressure under the conditionsdescribed in Study 1, with the exception of adjusting the spray gaspressure to 15 psi, the flow rate to 0.5 mL/min, the drying gas inlettemperature to 60° C., and the outlet temperature to 40° C. All fourstrains were titrated to 5.6 log ffu/mL and formulated with SD01 at 20%solids content. The solution pH was adjusted to 6.3.

Study 11—

Spray drying live measles virus. A live measles virus was low pressuresonically spray dried under the following conditions:

a) All formulations were prepared with Edmonton-Zagreb live attenuatedvirus cultured in Vero cells, which upon harvest had a fluorescencefocus assay (FFA) titer of 6.0 log₁₀. The viruses were stabilized in7.2% (w/v) sucrose and 50 mM potassium phosphate buffer. A series ofmeasles formulations were prepared by mixing the measles virus solutionwith a concentrated stock containing the various components as shown inTable 1. Formulated measles virus had a titer of 5.4 log₁₀.

b) The formulation was combined at a flow of 0.5 mL/min with a stream ofnitrogen gas at 15 psi in a mixing chamber of a nozzle.

c) The nozzle was vibrated at ultrasonic frequencies.

d) The formulation/gas mixture was sprayed into a drying chamber whiledrying gas flowed into the chamber at 60° C. Drying gas exited thechamber at 40° C. outlet temperature.

e) Dry particles were collected and the virus titer was analyzed (afterreconstitution) in order to determine the degree of process-relatedloss. The residual moisture content of the spray dried powder as well asthe titers of the reconstituted measles virus are shown in Table 2.

f) The collected powders were stored at 4, 25, and at 37° C. in order todetermine the stability of the spray dried measles virus. The virustiters upon reconstitution at various time points are shown in Table 2.

TABLE 1 Formulation components for measles virus comprised of 25% solidscontent with a virus titer of 5.4log₁₀. Formulation Components M1 M2 M3M4 M5 M6 M7 M8 Trehalose (%, w/v) 8.33 8.33 8.33 8.33 8.33 8.33 8.338.33 Sucrose (%, w/v) 16.7 12.7 6.4 6.7 7.4 10.4 9.4 16.7 Myo-inositol(%, w/v) 10 Sorbitol (%, w/v) 3 Gelatin (%, w/v) 6.25 6.30 6.25 6.25Potassium PO₄ (mM) 69.4 69.4 69.4 69.4 69.4 69.4 69.4 Sodium Citrate(mM) 50 L-Arginine (%, w/v) 4 4 Human albumin (%, w/v) 1 Glycerol (%)1.25 1.25 1.25 1.25 1.25 1.25 1.25 Pluronic F68 (%) 0.06 0.06 0.06 0.060.06 0.06 0.06 0.06 pH 7 7 7 7 7 7 7 7

TABLE 2 Stability of low pressure sonically spray dried measles virusstored at 37° C. Residual moisture contents of the spray dried measlesvirus are also provided. The measles virus titer prior to processing was5.4log TCID₅₀/mL. Moisture Reconstituted virus titer (log TCID₅₀/mL)after Formu- Content storage at 37° C. for the indicated time lation (%,w/w) Initial 1 week 2 weeks 4 weeks 8 weeks M1 3.7 5.4 4.1 3.7 3.7 2.8M2 3.9 5.2 4.2 3.8 3.6 3.5 M3 4.3 5.4 4.2 4.2 3.8 3.2 M4 3.5 5.8 4.2 4.33.7 2.5 M5 3.1 5.7 4.1 3.8 3.4 2.7 M6 3.6 4.9 4.1 3.8 3.8 3.0 M7 3.3 5.04.0 4.2 3.5 3.0 M8 3.2 5.4 3.8 3.8 3.8 2.9

Study 12—

Spray drying live measles virus. A live measles virus was low pressuresonically spray dried under the conditions described in Study 11, withthe exception of adjusting formulated measles virus titer to 4.3 log₁₀.

a) The formulation components used to stabilize the measles virus areshown in Table 3.

b) Dry particles were collected and the virus titer was analyzed (afterreconstitution) in order to determine the degree of process-relatedloss. The residual moisture content of the spray dried powder as well asthe titers of the reconstituted measles virus are shown in Table 4.

c) The dry measles particles were stored at 4, 25, and at 37° C. inorder to determine the stability of the spray dried powders. The virustiters upon reconstitution at the various time points are shown in Table4.

TABLE 3 Formulations for measles virus comprised of 25% solids contentwith a virus titer of 4.3log₁₀. Formulation Components M9 M10 M11 M12M13 M14 M15 M16 M17 Trehalose (%, w/v) 8.33 8.33 8.33 8.33 8.33 8.338.33 8.33 20.98 Sucrose (%, w/v) 12.7 12.65 12.65 12.65 12.65 12.6512.65 12.65 Potassium PO₄ (mM) 69.4 69.4 69.4 69.4 69.4 69.4 69.4 69.469.4 L-Arginine (%, w/v) 4 4 4 4 4 4 4 4 4 Glycerol (%) 1.25 1.25 1.251.25 1.25 1.25 1.25 1.25 Pluronic F68 (%) 0.06 0.06 0.06 0.06 0.06 0.060.06 0.06 0.06 MgCl₂ (mM) 2 CaCl₂ (mM) 2 2 ZnCl₂ (mM) 2 2 MnCl₂ (mM) 2EDTA (mM) 2 pH 7 6 6 6 6 6 6 6 6

TABLE 4 Stability of low pressure sonically spray dried measles virusstored at 37° C. Residual moisture contents of the spray dried measlesvirus are also provided. The measles virus titer prior to processing was4.3 log TCID₅₀/mL. Reconstituted virus titer (log Moisture TCID₅₀/mL)after storage at Content 37° C. for the indicated time Formulation (%,w/w) Initial 1 week 2 weeks 4 weeks M9 3.8 3.9 2.4 2.0 ND M10 3.6 3.72.8 2.5 ND M11 3.0 3.8 2.5 2.5 ND M12 3.3 3.6 2.5 2.5 ND M13 2.6 3.8 2.52.5 ND M14 2.8 3.6 2.7 2.7 2.3 M15 3.1 3.4 2.6 3.0 2.1 M16 3.6 3.9 3.32.9 2.5 M17 3.5 3.6 2.5 2.8 2.3 ND = not determined

Study 13—

Spray drying live Salmonella. A Salmonella Ty21a live formulation waslow pressure sonically spray dried under the following conditions:

a) Ty21a inocula were taken from frozen stock, thawed, and diluted 1:400in 50 ml of Brain Heart Infusion broth in 500 ml baffled Erlenmeyerflask. The culture was grown for 16 hours at 37° C., while being spun at240 rpm, to an OD of 2.17.

b) A formulated bacterial suspension was prepared by harvesting abacterial pellet by spinning the suspension for 10 minutes at 2500×g.Supernatant was discarded and the pellet was resuspended in the samevolume of formulation SD01 (2.5× above) to provide 25% solids.

c) Formulated material was titered at 3×10⁹ CFU/mL.

d) The formulation was combined at a flow of 0.75 mL/min with a streamof nitrogen gas at 60 psi in a mixing chamber of a nozzle.

e) The nozzle was vibrated at ultrasonic frequencies.

f) The formulation/gas mixture was sprayed into a drying chamber whiledrying gas flowed into the chamber at 45° C. Drying gas exited thechamber with a 35-37° C. outlet temperature.

g) Dry particles containing 2.2% residual moisture content werecollected. After reconstitution, the formulation had a time zero (t₀)titer of 5.7×10⁸ CFU/mL. See FIG. 6.

h) The dry Salmonella particles were stored at 25° C. in order todetermine the stability of the spray dried powders. The virus titersupon reconstitution at the various time points are shown in FIG. 7.

Study 14—

Low pressure sonic spraying of a bioactive protein. An alkalinephosphatase enzyme formulation was low pressure ultrasonically spraydried under the following conditions:

a) A solution of horse radish peroxidase (HRP), 356,000 AU/ml, wasprepared in liquid formulation SD01 comprised of 25% solids content.

b) The formulation was combined at a flow of 0.5 mL/min with a stream ofnitrogen gas at 60 psi in a mixing chamber of a nozzle.

c) The nozzle was vibrated at ultrasonic frequencies.

d) The formulation/gas mixture was sprayed into a drying chamber whiledrying gas flowed into the chamber at 45° C. Drying gas exited thechamber at 35-37° C. outlet temperature.

e) Dry particles containing 3.96% residual moisture content werecollected. Process-related loss was determined to be 0.06 log AU/ml uponreconstitution.

e) The dry HRP particles were stored at 25° C. in order to determine thestability of the spray dried powders. After 2 months of storage, the HRPmaintained an activity of 308,000 AU/ml.

Study 15—

Holding Rotavirus at high temperatures in organic solvent suspensions.Spray dried powder samples (approximately 800 mg) were suspended inapproximately 2 mL of various organic solvents and their mixtures (seeTable 5). Suspensions were placed in sample plates in a drying oven at55° C. for one hour. Samples were removed from the suspensions andallowed to dry for 15 minutes before measuring activity. The amount ofresidual solvent as well as the potency change due to solvent exposureare shown in Table 5.

TABLE 5 Residual solvent and potency upon suspension of spray driedRotavirus powder in indicated organic solvent. The suspension wasincubated for 1 hr at 55° C. followed by solvent evaporation. The spraydried Rotavirus powder had a titer of 5.8 ± 0.12 ffu/mL. ResidualPotency solvent Potency change Solvent (%) (log ffu/mL) (log ffu/mL)Chloroform 1.83 6.06 ± 0.03 0.26 Ethanol 7.46 5.04 ± 0.13 −0.76 Heptane4.91 5.97 ± 0.17 0.17 Isopropyl Alcohol 1.06 5.84 ± 0.12 0.04 MethylIsobutyl Ketone 2.55 5.78 ± 0.18 −0.02 Tetrahydrofuran 3.99 5.67 ± 0.06−0.13 Ethyl Acetate 5.02 5.77 ± 0.07 −0.03 Dichloromethane 1.35 5.73 ±0.23 −0.07 Dichloromethane:Ethanol:Isopropanol 2.83 5.83 ± 0.06 0.03(5:6:4)

Example 2 Narrow Particle Size Range from Viscous Solution

Study 1—

Spraying 10% sucrose at low pressures with the sonic nozzle. A 10%sucrose solution was sprayed using high frequency vibrations, resultingin the particle distribution shown in FIG. 2. The solution flowed intothe spray nozzle at a pressure of 20 psi.

The spray particle size distribution was obtained by analyzing the sprayplume produced by atomizing a 10% sucrose solution at 20 psi usingSpraytec. The particle size distribution is shown in Table 6 and theparticle size statistics are shown in Table 7.

Measurement values and settings included: open spray lens=300 mm, pathlength=100.0 mm, particulate refractive index=1.33+0.00i, scatterstart=1, dispersant refractive index=1.00, scatter end=35, particledensity=1.00 (gm/cc), scattering threshold=1, residual=2.75%, minimumsize=0.10 um, extinction analysis=off, and maximum size=2500.00 (um).

TABLE 6 Spray particle size distribution (See FIG. 2). Size (μm) % V < %V 0.117 0.00 0.00 0.136 0.00 0.00 0.158 0.00 0.00 0.185 0.00 0.00 0.2150.00 0.00 0.251 0.00 0.00 0.293 0.00 0.00 0.341 0.00 0.00 0.398 0.000.00 0.464 0.00 0.00 0.541 0.00 0.00 0.631 0.00 0.00 0.736 0.00 0.000.858 0.00 0.00 1.00 0.01 0.01 1.17 0.12 0.11 1.36 0.34 0.22 1.58 0.680.34 1.85 1.13 0.45 2.15 1.69 0.56 2.51 2.36 0.67 2.93 3.17 0.81 3.414.15 0.98 3.98 5.38 1.23 4.64 6.97 1.59 5.41 9.09 2.12 6.31 11.96 2.867.36 15.81 3.85 8.58 20.89 5.08 10.00 27.38 6.49 11.66 35.31 7.93 13.5944.50 9.19 15.85 54.49 9.99 18.48 64.57 10.08 21.54 73.87 9.30 25.1281.52 7.65 29.29 86.89 5.37 34.15 89.78 2.89 39.81 90.55 0.77 46.4290.55 0.00 54.12 90.55 0.00 63.10 90.55 0.00 73.56 90.55 0.00 85.7790.55 0.00 100.00 90.55 0.00 116.59 100.00 9.45 135.94 100.00 0.00158.49 100.00 0.00 184.79 100.00 0.00 215.44 100.00 0.00 251.19 100.000.00 292.87 100.00 0.00 341.46 100.00 0.00 398.11 100.00 0.00 464.16100.00 0.00 541.17 100.00 0.00 630.96 100.00 0.00 735.64 100.00 0.00857.70 100.00 0.00 1000.00 100.00 0.00

TABLE 7 Particle size statistics. Title Average σ Min Max Trans (%) 98.60.2139 97.8 98.9 Dv(10) (μm) 5.709 0.8944 4.552 8.543 Dv(50) (μm) 14.811.312 13.88 20.65 Dv(90) (μm) 34.94 111.8 30.21 441.7 % < 10μ (%) 27.384.349 13.93 32.86 D[4][3](μm) 23.45 12.95 22 90.02 D[3][2](μm) 10.631.127 9.38 14.7 Cv (PPM) 0.4348 0.0923 0.3328 0.9466 Span 1.975 7.4941.667 27.31 Dv(0) (μm) 0.8844 0.05117 0.8572 1.002 Standard Values:Trans = 98.6 (%) Cv = 0.4348 (PPM) SSA = 0.5645 (m²/cc) Dv(10) = 5.709(μm) Dv(50) = 14.81 (μm) Dv(90) = 34.94 (μm) Span = 1.975 D[3][2] =10.63 (μm) D[4][3] = 23.45 (μm) 153 Records Averaged

Study 2—

Spraying 10% sucrose at 30 psi with the sonic nozzle. A 10% sucrosesolution was sprayed using high frequency vibrations, resulting in theparticle distribution shown in FIG. 3.

The spray particle size distribution was obtained by analyzing the sprayplume produced by atomizing a 10% sucrose solution at 20 psi usingSpraytec. The particle size distribution is shown in Table 8 and theparticle size statistics are shown in Table 9. Measurement values andsettings included: open spray lens=300 mm, path length=100.0 mm,particulate refractive index=1.33+0.00i, scatter start=1, dispersantrefractive index=1.00, scatter end=35, particle density=1.00 (gm/cc),scattering threshold=1, residual=1.88%, minimum size=0.10 um, extinctionanalysis=off, and maximum size=2500.00 (um).

TABLE 8 Spray particle size distribution (See FIG. 3). Size (μm) % V < %V 0.117 0.00 0.00 0.136 0.00 0.00 0.158 0.00 0.00 0.185 0.00 0.00 0.2150.00 0.00 0.251 0.00 0.00 0.293 0.00 0.00 0.341 0.00 0.00 0.398 0.000.00 0.464 0.00 0.00 0.541 0.00 0.00 0.631 0.00 0.00 0.736 0.00 0.000.858 0.04 0.04 1.00 0.18 0.14 1.17 0.46 0.28 1.36 0.93 0.47 1.58 1.620.69 1.85 2.59 0.97 2.15 3.89 1.30 2.51 5.57 1.68 2.93 7.69 2.12 3.4110.30 2.61 3.98 13.44 3.14 4.64 17.16 3.72 5.41 21.49 4.33 6.31 26.424.93 7.36 31.93 5.51 8.58 37.93 6.00 10.00 44.30 6.36 11.66 50.81 6.5213.59 57.20 6.39 15.85 63.12 5.92 18.48 68.21 5.09 21.54 72.15 3.9425.12 74.74 2.59 29.29 75.98 1.24 34.15 76.15 0.17 39.81 76.15 0.0046.42 76.15 0.00 54.12 76.15 0.00 63.10 76.15 0.00 73.56 76.15 0.0085.77 76.15 0.00 100.00 76.15 0.00 116.59 76.15 0.00 135.94 76.15 0.00158.49 76.15 0.00 184.79 76.15 0.00 215.44 76.15 0.00 251.19 76.15 0.00292.87 76.15 0.00 341.46 76.30 0.15 398.11 77.54 1.24 464.16 81.19 3.65541.17 87.26 6.07 630.96 93.69 6.43 735.64 98.06 4.38 857.70 99.76 1.701000.00 100.00 0.24

TABLE 9 Particle Size Statistics. Title Average σ Min Max. Trans (%)98.0 0.3497 97.1 99.8 Dv(10) (μm) 3.36 0.4157 2.322 5.776 Dv(50) (μm)11.44 52.32 6.325 572.9 Dv(90) (μm) 576.3 68.01 14.39 682.6 % < 10μ (%)44.3 4.539 25.05 73.63 D[4][3] 141.7 40.36 7.657 393.8 D[3][2] 7.8460.641 4.781 11.82 Cv (PPM) 0.4526 0.1244 0.03584 0.6959 Span 50.09 9.9311.141 65.14 Dv(0) (μm) 0.7449 0.04204 0.734 1.001 Standard Values: Trans= 98.0 (%) Cv = 0.4526 (PPM) SSA = 0.7647 (m²/cc) Dv(10) = 3.36 (μm)Dv(50) = 11.44 (μm) Dv(90) = 576.3 (μm) Span = 50.09 D[3][2] = 7.846(μm) D[4][3] = 141.7 (μm) 177 Records Averaged

Study 3—

Spraying water at low pressures with the sonic nozzle. Water was sprayedusing high frequency vibrations, resulting in the particle distributionshown in FIG. 4. Water flowed into the spray nozzle at a pressure of 20psi.

The spray particle size distribution was obtained by analyzing the sprayplume produced by atomizing a 10% sucrose solution at 20 psi usingSpraytec. The particle size distribution is shown in Table 10 and theparticle size statistics are shown in Table 11. Measurement values andsettings included: open spray lens=300 mm, path length=100.0 mm,particulate refractive index=1.33+0.00i, scatter start=1, dispersantrefractive index=1.00, scatter end=36, particle density=1.00 (gm/cc),scattering threshold=1, residual=2.92%, minimum size=0.10 um, extinctionanalysis=off, and maximum size=2500.00 (um).

TABLE 10 Spray particle size distribution for water (See FIG. 4). Size(μm) % V < % V 0.117 0.00 0.00 0.136 0.00 0.00 0.158 0.00 0.00 0.1850.00 0.00 0.215 0.00 0.00 0.251 0.00 0.00 0.293 0.00 0.00 0.341 0.000.00 0.398 0.00 0.00 0.464 0.00 0.00 0.541 0.00 0.00 0.631 0.00 0.000.736 0.00 0.00 0.858 0.00 0.00 1.00 0.06 0.06 1.17 0.19 0.14 1.36 0.420.22 1.58 0.72 0.30 1.85 1.08 0.36 2.15 1.50 0.41 2.51 1.96 0.47 2.932.50 0.54 3.41 3.17 0.66 3.98 4.04 0.87 4.64 5.26 1.22 5.41 7.05 1.796.31 9.67 2.62 7.36 13.44 3.78 8.58 18.69 5.25 10.00 25.62 6.93 11.6634.26 8.64 13.59 44.32 10.05 15.85 55.15 10.83 18.48 65.82 10.67 21.5475.28 9.46 25.12 82.60 7.32 29.29 87.29 4.69 34.15 89.45 2.16 39.8189.80 0.34 46.42 89.80 0.00 54.12 89.80 0.00 63.10 89.80 0.00 73.5689.80 0.00 85.77 89.80 0.00 100.00 89.80 0.00 116.59 99.59 9.79 135.94100.00 0.41 158.49 100.00 0.00 184.79 100.00 0.00 215.44 100.00 0.00251.19 100.00 0.00 292.87 100.00 0.00 341.46 100.00 0.00 398.11 100.000.00 464.16 100.00 0.00 541.17 100.00 0.00 630.96 100.00 0.00 735.64100.00 0.00 857.70 100.00 0.00 1000.00 100.00 0.00

TABLE 11 Particle size statistics. Title Average σ Min Max Trans (%)97.3 0.205 96.5 97.6 Dv(10) (μm) 6.408 0.7384 5.659 8.579 Dv(50) (μm)14.74 1.198 14.16 19.27 Dv(90) (μm) 100.3 149.3 31.83 397 % < 10μ (%)25.63 3.825 14.15 29.01 D[4][3] (μm) 24.09 14.54 24.16 74.82 D[3][2](μm) 11.13 0.9259 10.39 14.01 Cv (PPM) 0.8811 0.1327 0.7427 1.414 Span6.371 10.14 1.772 26.19 Dv(0) (μm) 0.8607 0.05319 0.7463 0.8814 StandardValues: Trans = 97.3 (%) Cv = 0.8811 (PPM) SSA = 0.5390 (m²/cc) Dv(10) =6.408 (μm) Dv(50) = 14.74 (μm) Dv(90) = 100.3 (μm) Span = 6.371 D[3][2]= 11.13 (μm) D[4][3] = 24.09 (μm) 446 Records Averaged

Study 4—

Spraying water, 10% sucrose, 20% sucrose, and SD01 (20%) at lowpressures with the sonic nozzle. Water, 10% sucrose, 20% sucrose, andSD01 (20%) were sprayed using high frequency vibrations at flow ratesranging from 2-4 mL/min at spray nozzle pressures ranging from 4-21 psi.

The spray particle size distribution was obtained by analyzing the sprayplume produced by the sonic nozzle using Spraytec. The particle sizedistributions, more specifically D_(v)10, D_(v)50, and D_(v)90, areshown in Tables 12-14 respectively.

TABLE 12 Particle size statistics (D_(v)10) of water, 10% sucrose, 20%sucrose, and 20% SD01 at various flow rates and spray nozzle pressures.Average and standard deviation values (in μm) were calculated fromapproximately 500 measurements. 10% 20% SD01 P (psi) Water sucrosesucrose (20%) q = 2 mL/min 4  19 ± 1.2 14.9 ± 0.8 13.0 ± 0.8 13.5 ± 1.010 9.7 ± 0.2  8.4 ± 0.2  8.1 ± 0.2  8.0 ± 0.2 21 8.0 ± 0.1  3.0 ± 0.1 7.0 ± 0.1  6.0 ± 0.2 q = 0.3 mL/min 4 16.8 ± 1.1 15.5 ± 0.9 12.5 ± 1.014.8 ± 0.9 10  9.8 ± 0.2  8.8 ± 0.2  8.5 ± 0.3  8.5 ± 0.4 21  8.1 ± 0.1 7.2 ± 0.2  7.2 ± 0.2  6.3 ± 0.3 q = 4 mL/min 4 18.5 ± 0.9 17.2 ± 0.815.8 ± 1.4 18.0 ± 1.1 10 10.0 ± 0.2  9.0 ± 0.2  8.2 ± 0.2  9.0 ± 0.2 21 8.1 ± 0.1  7.5 ± 0.2  7.4 ± 0.2  7.2 ± 0.2

TABLE 13 Particle size statistics (D_(v)50) of water, 10% sucrose, 20%sucrose, and 20% SD01 at various flow rates and spray nozzle pressures.Average and standard deviation values (in μm) were calculated fromapproximately 500 measurements. 10% 20% SD01 P (psi) Water sucrosesucrose (20%) q = 2 mL/min 4 40.1 ± 10.0 32.5 ± 2.5 28.5 ± 1.5 31.2 ±4.4 10 20.0 ± 0.5  17.5 ± 0.4 15.0 ± 1.6 13.0 ± 1.1 21 9.5 ± 0.1  7.7 ±0.1  7.2 ± 0.0  7.6 ± 0.1 q = 3 mL/min 4 32.5 ± 2.7 34.2 ± 3.2 30.5 ±2.2 33.0 ± 4.5 10 20.5 ± 0.4 18.0 ± 0.3 17.5 ± 0.7 16.2 ± 0.9 21 10.0 ±0.1  7.9 ± 0.1  7.7 ± 0.1  7.8 ± 0.1 q = 4 mL/min 4 36.0 ± 4.0 40.0 ±6.2 37.5 ± 5.3 43.0 ± 8.0 10 22.0 ± 0.2 19.5 ± 1.3 18.5 ± 0.7 17.8 ± 0.621 10.0 ± 0.1  8.1 ± 0.1  7.9 ± 0.1  8.2 ± 0.1

TABLE 14 Particle size statistics (D_(v)90) of water, 10% sucrose, 20%sucrose, and 20% SD01 at various flow rates and spray nozzle pressures.Average and standard deviation values (in μm) were calculated fromapproximately 500 measurements. 10% 20% SD01 P (psi) Water sucrosesucrose (20%) q = 2 mL/min 4 120.4 ± 25.1 85.1 ± 15.4 74.0 ± 15.6 78.0 ±20.7 10 35.0 ± 8.0 35.0 ± 3.1  38.4 ± 3.8  32.2 ± 4.1  21 22.4 ± 1.122.0 ± 2.1  18.3 ± 2.0  15.0 ± 2.2  q = 3 mL/min 4 98.7 ± 15.1 105.1 ±20.4 92.1 ± 16.5 102.2 ± 18.1 10 37.0 ± 5.2  38.4 ± 3.0 41.8 ± 3.2  35.0± 5.4 21 22.2 ± 1.0  22.3 ± 1.0 20.0 ± 1.2  17.1 ± 1.5 q = 4 mL/min 4116.3 ± 20.2 124.1 ± 22.1 122.5 ± 18.1 125.4 ± 21.2 10 43.4 ± 8.2 42.1 ±4.2 41.0 ± 3.1 40.4 ± 3.1 21 21.0 ± 1.7 24.2 ± 1.0 21.2 ± 1.0 21.2 ± 1.3

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from reading this disclosure that various changes in form anddetail can be made without departing from the true scope of theinvention. For example, many of the techniques and apparatus describedabove can be used in various combinations.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

What is claimed is:
 1. A method of preparing a bioactive material fororal delivery, the method comprising: preparing a suspension or solutioncomprising the bioactive material and a polymer; forming a mixture ofpressurized gas with the solution or suspension within a nozzle;vibrating the nozzle at a frequency ranging from 1 kHz to 100 kHz, whilespraying the mixture to form a gaseous suspension of droplets; dryingthe droplets in a stream of drying gas to form powder particles of thebioactive material and, incorporating the particles into a dosage formatfor oral delivery.
 2. The method of claim 1, wherein the bioactivematerial is selected from the group consisting of bioactive proteins,peptides, antibodies, monoclonal antibodies, enzymes, serums, vaccines,nucleic acids, bacteria, prokaryotic cells, eukaryotic cells, liposomes,Pneumococcus, Francisella tularensis, Mycobacterium, Salmonella,Shigella, Listeria, Pseudomonas, Staphylococcus, Streptococcus, E. coli,and a virus.
 3. The method of claim 2, wherein the virus is selectedfrom the list consisting of: rotavirus, adenovirus, measles virus, mumpsvirus, rubella virus, polio virus, influenza virus, parainfluenza virus,respiratory syncytial virus, herpes simplex virus, SARS virus, coronavirus family members, cytomegalovirus, human metapneumovirus, filovirus,and Epstein-Bar virus.
 4. The method of claim 1, wherein a pressure ofthe mixture ranges from 10 psi to 100 psi.
 5. The method of claim 1,further comprising heating the nozzle to reduce the viscosity of thesuspension or solution as it is vibrated.
 6. The method of claim 1,wherein the solution or suspension further comprises a polyol selectedfrom the group consisting of: sucrose, trehalose, glucose, raffinose,sorbose, melezitose, glycerol, fructose, mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, galactose, glucose, mannitol,xylitol, erythritol, threitol, dextrose, fucose, trehalose, polyasparticacid, inositol hexaphosphate (phytic acid), sialic acid,N-acetylneuraminic acid-lactose, and sorbitol.
 7. The method of claim 1,wherein the polymer is selected from the group consisting of: polyvinylpyrrolidone (PVP), gelatin, collagen, chondroitin sulfate, starch,starch derivatives, carboxymethyl starch, hydroxyethyl starch (HES),polyvinyl alcohol, and dextran.
 8. The method of claim 1, wherein thesuspension or solution further comprises a surfactant selected from thegroup consisting of: block co-polymers of polyethylene and polypropyleneglycol, polyethylene glycol sorbitan monolaurate, and polyoxyethylenesorbitan monooleate.
 9. The method of claim 1, wherein the suspension orsolution further comprises an amino acid selected from the groupconsisting of: arginine, alanine, lysine, methionine, histidine, andglutamic acid.
 10. The method of claim 1, wherein the suspension orsolution further comprises a divalent cation selected from the groupconsisting of Ca²⁺, Zn²⁺, Mg²⁺, and Mn²⁺.
 11. The method of claim 1,wherein the pressurized gas is selected from the group consisting of:air, nitrogen, oxygen, helium, carbon dioxide, sulfur hexafluoride,chlorofluorocarbons, methane, fluorocarbons, nitrous oxide, xenon,propane, n-pentane, ethanol, and water.
 12. The method of claim 1,wherein said forming of the mixture comprises flowing the suspensioninto the nozzle at a pressure less than 10 psi and flowing the gas intothe nozzle at a pressure between 10 psi and 75 psi.
 13. The method ofclaim 1, wherein the gas contains a modifier selected from the groupconsisting of: methanol, ethanol, isopropanol, chloroform, heptane,methyl isobutyl ketone, tetrahydrofuran, ethyl acetate, dichloromethane,dichloromethane:ethanol:isopropanol (5:6:4), and acetone.
 14. The methodof claim 1, wherein said forming of the mixture comprises flowing thesolution or suspension with the pressurized gas through a mixingchamber.
 15. The method of claim 1, wherein said mixing occurs beforesaid spraying.
 16. The method of claim 1, wherein the vibrations aregenerated using the pressurized gas.
 17. The method of claim 1, whereinthe frequency comprises a frequency ranging from more than 10 kHz toabout 30 kHz.
 18. The method of claim 1, wherein the vibrations occurwhile the solution or suspension and the gas are within the nozzle. 19.The method of claim 1, further comprising suspending the powderparticles in an organic solvent.
 20. The method of claim 19, whereinincorporating the particles into a dosage format comprises drying thesolvent to form a solid material comprising the bioactive material. 21.The method of claim 1, wherein the dosage form comprises a wafer, a thinfilm, a capsule, or a tablet.
 22. The method of claim 1, wherein theformulation comprises constituents other than a carbonate.
 23. Themethod of claim 1, wherein the formulation comprises total solidsranging from 20% to 50% by weight.
 24. The method of claim 1, whereinthe spraying is adapted to provide droplets range in size from about 8μm to about 43 μm.
 25. The particles in the oral dosage format preparedby the method of claim
 21. 26. The particles in the oral dosage formatprepared by the method of claim 19.